marine drugs

Review

An Overview of the Medical Applications of MarineSkeletal Matrix ProteinsM. Azizur Rahman 1,2

1 Department of Chemical & Physical Sciences, University of Toronto Mississauga,Mississauga, ON L5L 1C6, Canada; mazizur.rahman@utoronto.ca or azizurr142@gmail.com;Tel.: +1-647-892-4221

2 HiGarden Inc., Markham, ON L3R 3W4, Canada

Academic Editor: Se-Kwon KimReceived: 9 May 2016; Accepted: 7 September 2016; Published: 12 September 2016

Abstract: In recent years, the medicinal potential of marine organisms has attracted increasingattention. This is due to their immense diversity and adaptation to unique ecological niches that hasled to vast physiological and biochemical diversification. Among these organisms, marine calcifiersare an abundant source of novel proteins and chemical entities that can be used for drug discovery.Studies of the skeletal organic matrix proteins of marine calcifiers have focused on biomedicalapplications such as the identification of growth inducing proteins that can be used for boneregeneration, for example, 2/4 bone morphogenic proteins (BMP). Although a few reports on thefunctions of proteins derived from marine calcifiers can be found in the literature, marine calcifiersthemselves remain an untapped source of proteins for the development of innovative pharmaceuticals.Following an overview of the current knowledge of skeletal organic matrix proteins from marinecalcifiers, this review will focus on various aspects of marine skeletal protein research includingsources, biosynthesis, structures, and possible strategies for chemical or physical modification.Special attention will be given to potential medical applications and recent discoveries of skeletalproteins and polysaccharides with biologically appealing characteristics. In addition, I will introducean effective protocol for sample preparation and protein purification that includes isolation technologyfor biopolymers (of both soluble and insoluble organic matrices) from coralline algae. These algaeare a widespread but poorly studied group of shallow marine calcifiers that have great potential formarine drug discovery.

Keywords: biomineralization; coralline algae; chitin; collagen; marine calcifiers; marine skeletalproteins; proteomics

1. Introduction

Skeletal proteins and polysaccharides in marine organisms are present as complex mixtureswithin organic matrices. The organic matrices of marine calcifiers, for example, are a potentiallyuntapped source of skeletal proteins [1–6]. Organic matrices have the advantage of being naturallyproduced, retaining the native, functional conformation of the original proteins. Moreover, a significantnumber of calcifying marine invertebrates produce polysaccharides within their extracellularmatrices and connective tissues [7,8] that have molecular structures and functions similar to humanversions [1,6]. Polysaccharides derived from marine invertebrate extracellular matrices encompass anenormous variety of structures and should be considered as an extraordinary source of biochemicaldiversity. However, they remain largely under-exploited with respect their potential in medicalapplications [9,10]. Macromolecules derived from marine calcifiers that hold promise for biomedicalapplications include a broad range of protein and sugar (carbohydrates and lectins) molecules thatparticipate in signaling, development, regeneration, and metabolism.

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It is has been hypothesized that marine skeletal proteins that function in biomineral growth,maintenance, and repair could facilitate tissue engineering. For example, some of these proteins withhuman physiological activity can help accelerate lab-based bone morphogenesis and increase bonevolumes with efficacies equivalent to currently used recombinant proteins [1]. Proteins with potentialfor bone repair and drug discovery, extracted either from naturally occurring skeletal organic matricesor derived from cultivated tissues, can be identified and isolated using chromatography, cell assays andproteomic methods [1,9,11]. Proteomics is a high-throughput analytical method for rapidly identifyingknown or unknown proteins in complex mixtures [5]. If purification methods can be establishedfor skeletal proteins derived from calcifying marine organisms, researchers in the emerging fields ofproteomics and medicinal chemistry could utilize these methods for subsequent drug discovery and,as a more specific example, bone repair. Currently, primary sequences of different skeletal proteinsfrom marine organisms are available in public databases, and this information can be used to infer thebiological function and origin of individual proteins and provide clues related to the mechanisms offormation of any skeleton.

Pharmaceutical industries now accept the world’s oceans as a major frontier for medical research.The emergence of this relatively new area of scientific exploration has been of enormous interest to thepopular and scientific press, and several review publications have appeared on the topic [1,9,11]. In thereview presented here, we focus on recent progress in the discovery and production of new marineskeletal proteins and polysaccharides of pharmaceutical interest. We also introduce a new technique forpurifying compounds derived from the skeletal organic matrices of coralline algae that will be usefulfor proteomic analysis and purifying biopolymers such as chitin and collagen. Overall, this reviewdemonstrates the existence of unique biomineralization-related skeletal proteins in marine calcifiersthat hold promise for drug development, and moreover, provides the first description of proteinaceouscomponents in coralline red algae.

2. Applications and Modification Strategies of Marine Skeletal Proteins for Drug Discovery

Marine calcifiers (shallow, mid-shelf, and deep sea) are widespread in oceans globally. However,due to the lack of effective extraction/analytical methods, the applications of these potential resourcesfor drugs are comparatively fewer than for other marine organisms. Recently, we perceived proteininduced crystallization [2,7,8,12,13], which showed potential crystal design and growth that could helpmedicinal chemistry in drug design. Our primary chemical proteomic results from soft coral revealeda number of molecules with high concentrations [5,14]. In addition, some proteins extracted from softcorals are homologous with many human proteins, making them useful due to their similarity [15,16].The information with respect to the close homology of soft coral and human proteins provides usfunctional and evolutionary clues on the structure and functions of their sequences. These homologousproteins could lead to possible drug discovery and form a potential resource for biotechnologicalresearch. It is our hope that further sequence studies of these materials will contribute to a betterunderstanding of structural proteins in soft corals. Bioassay-directed fractionation of octocoralCespitularia hypotentaculata, which has a novel endoskeleton, yielded the diterpene cespitularinA–D, the norditerpene cespitularin E and three other diterpenes, cespitularin F–H [17]. Two newdolabellane-type diterpenoids and the known diterpene clavenone [18] were isolated from a octocoralClavularia species [19]. A saponin compound was isolated from the octocoral Lobophytum spp.,which was collected from Hainan Island, China.

Among the marine calcifiers, very few scleractinian corals were investigated. In a recent review,the authors discussed the potential of scleractinian coral, which has therapeutic characteristics,including anti-inflammatory properties, anticancer properties, bone repair, and neurological benefits [6].Research on the scleractinian coral Montipora spp. from the republic of Korea (South Korea)found three diacetylenes (1, 4, 6). One of these was a potent cytotoxin with respect to a rangeof tumor cell lines [20]. The authors tested the extracted compounds against a panel of humancancer cell lines and the structures have been interpreted on the basis of spectroscopic evidence.

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These three compounds showed a structural activity profile to similar to those previously reported [21].The results showed that the compound 6 with b-hydroxy ketone functionality has strong cytotoxicproperties and Methyl montiporate C (1) was active only against a skin cancer cell line, whilecompound 4 was moderately active. Extracts from the calcifying octocorals Pseudopterogorgia elizabethae(which contains pseudopterosins) and Eunicea fusca (which contains fucoside-A) can be used inthe cosmetic industry [22]. Similarly, coral (endoskeletons and exoskeletons) and coralline algalskeletons could be used for cosmetics as both contain a high concentration of organic matrixcomponents [7,13,23].

In recent years, numerous applications have been proposed for chitosan-based deliverydevices [24–26], however, most of these were unrelated to marine calcifiers. Chitosan is a copolymer ofβ-(1-4)-linked 2-acetamido-2-deoxy-D-glucopyranose and 2-amino-2-deoxy-D-glucopyranose, obtained bydeacethylation of the naturally occurring chitin. Chitin was firstly extracted from the exoskeleton ofmarine organisms, mainly crabs and shrimps, as described by Burrows [27]. This polymer has alsorecently been extracted from coralline algae [7], which opened the doors for possible applicationsof these biomaterials using a group of marine calcifers which are found in shallow water and areeasy-to-collect, abundant and widespread. The major applications of chitosan are for biomaterials,pharmaceuticals, foodstuff treatment (e.g., flocculation, clarification, etc., due to its efficient interactionwith other polyelectrolytes), cosmetics, metal ion sequestration, and agriculture [28–31]. Development ofchitosan chemistry has relevant biomedical applications, particularly in the field of drug delivery [32].While chitin is insoluble in most common solvents, chitosan can be readily turned into fibers aswell as films, or triggered in a variety of micromorphologies from its acidic aqueous solutions.Protein-polysaccharides play an important role in biomedical and pharmaceutical applications.However, at times the properties of such biomaterials do not meet the needs for exact applications.As a result, approaches that chemically or physically modify their structure and, thus, physical-chemicalproperties are increasingly gaining interest [33,34]. With respect to the polysaccharides’ chitin andchitosan, it is possible to target the reaction using sulfur trioxide-pyridine at two sites or at onlyone specific site, following different pathways of synthesis [35]. Great efforts have thus focusedon the progress of efficient modification reactions in well-controlled conditions under tolerabletemperatures [35]. For example, modification reactions of water-soluble chitin can be conductedin aqueous solutions or in organic solvents in an engorged state under mild conditions, and selectiveN-acetylation [35]. Some significant chemical reactions of acylation, alkylation, Schiff base formationand reductive N-alkylation, carboxyalkylation, N-phthaloylation are well described [35].

3. A Promising Future for Marine Calcifiers in Drug Discovery

Marine resources such as coral, mollusk and coralline algae could be a major source of medicinesover the next decades. It is estimated that marine ecosystems, such as those found in coral reefs or ata deep sea level have greater biological diversity than those of tropical rain forests. However, as withtropical rain forests, coral reefs represent considerable untouched potential in the science of medicine.

At present, marine calcifier collection and drug appraisal occurs successfully. However, there isno question that these resources are inadequate and it is possible that collectable marine organisms willbe almost completely explored within the next 20 years. There is still a doubt as to where scientists willturn in order to ensure a continuing flow of new medicines. The solution is difficult, however drugscan now be developed using many methods such as computer-aided design, combinatorial synthesisand proteomics. The chemical multiplicity of marine ecosystems, from simple to complex peptideand protein extraction, draws us in the direction of the discovery of new marine natural products invarious therapeutic areas such as cancer, inflammation, microbial infections, and various other deadlydiseases [36]. Cancer is the biggest challenge of the current century, and marine calcifying organismsshow new promise in fighting against this and other dangerous diseases.

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4. A Novel Approach to Isolation, Purification and Characterization of Marine Skeletal Proteins

Isolation and purification of skeletal proteins from marine calcifiers are complex because ofthe potential for contamination of the soft tissues and the high sensitivity of organic matrices tohandling. However, successfully purified skeletal proteins from several groups of marine calcifiershave recently emerged [4,5,14,22,23,37]. The overview concerning marine skeletal proteins presentedabove allows us to understand some newly developed techniques [5,12,14–16,23,38–40] as well asuseful methods for isolating and purifying skeletal proteins and proteomic analysis. Among marinecalcifiers, we recently investigated coralline red algae, which has specific biological characteristics [7]and contains high concentrations of soluble organic matrix (SOM) and insoluble organic matrix (IOM)fractions. High concentrations of both chitin and collagen biopolymers are present in SOM and IOM(Figure 1). Coralline algal concentrations of SOM (0.9%) and IOM (4.5%) are significantly higherthan those of other skeletal marine calcifiers such as octocorals, with SOM and IOM concentrationsof 0.03% and 0.05%, respectively [5,13,15]. The highly concentrated biopolymers present in skeletalorganic matrices open up the possibility for future drug development, because these two polymers arefrequently utilized in drug design [24,29–31,41–46].

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recently emerged [4,5,14,22,23,37]. The overview concerning marine skeletal proteins presented

above allows us to understand some newly developed techniques [5,12,14–16,23,38–40] as well as

useful methods for isolating and purifying skeletal proteins and proteomic analysis. Among marine

calcifiers, we recently investigated coralline red algae, which has specific biological characteristics [7]

and contains high concentrations of soluble organic matrix (SOM) and insoluble organic matrix

(IOM) fractions. High concentrations of both chitin and collagen biopolymers are present in SOM and

IOM (Figure 1). Coralline algal concentrations of SOM (0.9%) and IOM (4.5%) are significantly higher

than those of other skeletal marine calcifiers such as octocorals, with SOM and IOM concentrations

of 0.03% and 0.05%, respectively [5,13,15]. The highly concentrated biopolymers present in skeletal

organic matrices open up the possibility for future drug development, because these two polymers

are frequently utilized in drug design [24,29–31,41–46].

Figure 1. Identification of chitin and collagen in algal skeletal protein-polysaccharides complexes.

Structural comparison of FTIR spectra between organic matrix fractions (soluble organic matrix

(SOM) and insoluble organic matrix (IOM)) and bulk skeletal powder. Graphs for SOM, IOM fractions

and bulk skeletal powder are indicated. Different colored boxes in the spectra indicate involvement

of molecules in SOM and IOM fractions in forming skeletal structure in coralline algal calcification

system. (Reproduced from Scientific Reports, Rahman and Halfar 2014 [7]).

Detailed geochemical studies of coralline algae [47–51] provide a broad spectrum of

environmental and structural background information. However, there is a lack of information on

the protein-polysaccharide complex in the coralline algal skeleton, which plays a key role in the

regulation of biocalcification [7] and may contain prospective biomaterials for drug development.

Hence, we have developed a useful technique from sample preparation to protein isolation for the

Sub-Arctic coralline alga Clathromorphum compactum (Figure 2, see Ref. [7] for details) using recently

developed analytical approaches for other marine calcifiers (Figure 2, Refs. [5,12,23]). We

characterized the SOM-polysaccharide complex from its CaCO3 skeleton, which is involved in the

biocalcification process. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)

analysis [52] of the preparations [5,14,23] showed two bands of proteins with molecular masses of

250-kDa and 30-kDa (Figure 3A, lane 1 and 2). The protein with molecular masses of 30-kDa was by

far the most abundant protein, whereas the 250 kDa protein band was weak and somewhat faint

Figure 1. Identification of chitin and collagen in algal skeletal protein-polysaccharides complexes.Structural comparison of FTIR spectra between organic matrix fractions (soluble organic matrix (SOM)and insoluble organic matrix (IOM)) and bulk skeletal powder. Graphs for SOM, IOM fractions andbulk skeletal powder are indicated. Different colored boxes in the spectra indicate involvement ofmolecules in SOM and IOM fractions in forming skeletal structure in coralline algal calcification system.(Reproduced from Scientific Reports, Rahman and Halfar 2014 [7]).

Detailed geochemical studies of coralline algae [47–51] provide a broad spectrum ofenvironmental and structural background information. However, there is a lack of information onthe protein-polysaccharide complex in the coralline algal skeleton, which plays a key role in theregulation of biocalcification [7] and may contain prospective biomaterials for drug development.Hence, we have developed a useful technique from sample preparation to protein isolation forthe Sub-Arctic coralline alga Clathromorphum compactum (Figure 2, see Ref. [7] for details) usingrecently developed analytical approaches for other marine calcifiers (Figure 2, References [5,12,23]).We characterized the SOM-polysaccharide complex from its CaCO3 skeleton, which is involved inthe biocalcification process. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)

Mar. Drugs 2016, 14, 167 5 of 9

analysis [52] of the preparations [5,14,23] showed two bands of proteins with molecular masses of250-kDa and 30-kDa (Figure 3A, lane 1 and 2). The protein with molecular masses of 30-kDa wasby far the most abundant protein, whereas the 250 kDa protein band was weak and somewhatfaint (Figure 3A, lane 2). Periodic acid-Schiff (PAS) staining was used to identify chitin associatedglycoproteins. Interestingly, the 250-kDa protein was identified with high abundance as the onlyglycoprotein contained in the skeleton (Figure 3B, lane 1 and 2), even though it only appeared asa weak band in Coomassie Brilliant Blue (CBB) staining solution (Figure 3A). Chitin is the maincomponent of the protein-polysaccharide complex of cell walls [7,53,54]), which is also composed ofglycoprotein [55]. Protein-polysaccharide complexes are also present in coralline algal cell structures [7].Therefore, detection of a strong glycosylation protein in coralline algal skeletons reveals the presenceof highly abundant chitin. The chitin found in coralline alga has been recognized to be involved in thecalcification process [7] and this polymer is considered highly useful for drug design [24,29–31,41–46].Our observations therefore strongly suggest that the skeletal matrix proteins in coralline alga are notonly a structural protein but also have potential for drug development.

Mar. Drugs 2016, 14, 167  5 of 8 

glycoprotein contained in the skeleton (Figure 3B, lane 1 and 2), even though it only appeared as a 

weak  band  in Coomassie  Brilliant  Blue  (CBB)  staining  solution  (Figure  3A). Chitin  is  the main 

component of the protein‐polysaccharide complex of cell walls [7,53,54]), which is also composed of 

glycoprotein [55]. Protein‐polysaccharide complexes are also present in coralline algal cell structures 

[7]. Therefore, detection of  a  strong glycosylation protein  in  coralline  algal  skeletons  reveals  the 

presence of highly abundant  chitin. The  chitin  found  in  coralline alga has been  recognized  to be 

involved in the calcification process [7] and this polymer is considered highly useful for drug design 

[24,29–31,41–46]. Our observations  therefore  strongly  suggest  that  the  skeletal matrix proteins  in 

coralline alga are not only a structural protein but also have potential for drug development. 

 

Figure  2. Model  of  general  strategy  for  analyzing protein‐polysaccharides  complex  from  skeletal 

organic matrix of marine calcifiers. Figure 2. Model of general strategy for analyzing protein-polysaccharides complex from skeletalorganic matrix of marine calcifiers.

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Figure 3. Electrophoretic analysis of skeletal matrix proteins extracted  from  the coralline  red alga   

C.  compactum.  (A)  SDS‐PAGE  fractionation  with  Coomassie  Brilliant  Blue  (CBB)  staining  after 

purification of the skeletal proteins. Lane 1 and 2 indicate purified skeletal proteins. Arrows indicate 

protein bands; (B) SDS‐PAGE gel with Periodic Acid‐Schiff (PAS) staining to identify glycoprotein in 

skeletal  matrix  proteins  of  C.  compactum.  Lane  1  and  2,  a  strong  abundant  chitin  associated 

glycoprotein was identified (indicated by arrow) by periodic Acid‐Schiff staining. An eluate (derived 

from 5 g of algal skeleton) was run on 12% polyacrylamide gel M, protein marker. The Precision Plus 

SDS‐PAGE standard (Bio‐Rad) was used as protein marker for electrophoresis. 

5. Conclusions 

In  this  brief  review,  recent  advances  in  applications  of  protein‐polysaccharides  of marine 

calcifiers in the medical and pharmaceutical fields have been discussed. The results demonstrate the 

potential for marine calcifiers to generate new drugs. Understanding the proteinaceous components 

of marine calcifiers is an important step toward advancing the science of marine medicinal chemistry. 

Among the different sources of polysaccharides, algal polysaccharides such as chitin and collagen 

could play an  important role in future development of tissue engineering, bone regeneration, and 

much more. In light of these emerging findings, in the near future established techniques may also 

be potentially useful for isolating skeletal proteins from similar marine calcifiers for drug discovery. 

As a discovery‐driven science, the techniques discussed here allow researcher to identify candidate 

proteins  for  drug  discovery  and  identify  unknowns without missing  unanticipated  interactions. 

These techniques can be employed to dramatically improve the range of applications within the field 

of marine drug discovery. Since  the marine  realm consists of diverse ecosystems and matrices  in 

which these proteins reside, the development of effective methods for accessing proteins will be a 

continuing challenge in future years. 

Acknowledgments: The author thanks Jochen Halfar for providing coralline samples. The author also thanks 

Steven Short for providing electrophoresis apparatus. 

Conflicts of Interest: The author declares no conflict of interest. 

References 

1. Green, D.W.; Padula, M.P.; Santos, J.; Chou, J.; Milthorpe, B.; Ben‐Nissan, B. A therapeutic potential for 

marine skeletal proteins in bone regeneration. Mar. Drugs 2013, 11, 1203–1220. 

2. Rahman, M.A.; Fujimura, H.; Shinjo, R.; Oomori, T. Extracellular matrix protein in calcified endoskeleton: 

A potential additive for crystal growth and design. J. Cryst. Growth 2011, 324, 177–183. 

3. Rahman, M.A.;  Isa, Y.; Uehara, T. Proteins of calcified endoskeleton:  Ii partial amino acid sequences of 

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Figure 3. Electrophoretic analysis of skeletal matrix proteins extracted from the coralline red algaC. compactum. (A) SDS-PAGE fractionation with Coomassie Brilliant Blue (CBB) staining afterpurification of the skeletal proteins. Lane 1 and 2 indicate purified skeletal proteins. Arrows indicateprotein bands; (B) SDS-PAGE gel with Periodic Acid-Schiff (PAS) staining to identify glycoprotein inskeletal matrix proteins of C. compactum. Lane 1 and 2, a strong abundant chitin associated glycoproteinwas identified (indicated by arrow) by periodic Acid-Schiff staining. An eluate (derived from 5 g ofalgal skeleton) was run on 12% polyacrylamide gel M, protein marker. The Precision Plus SDS-PAGEstandard (Bio-Rad) was used as protein marker for electrophoresis.

5. Conclusions

In this brief review, recent advances in applications of protein-polysaccharides of marine calcifiersin the medical and pharmaceutical fields have been discussed. The results demonstrate the potentialfor marine calcifiers to generate new drugs. Understanding the proteinaceous components of marinecalcifiers is an important step toward advancing the science of marine medicinal chemistry. Among thedifferent sources of polysaccharides, algal polysaccharides such as chitin and collagen could play animportant role in future development of tissue engineering, bone regeneration, and much more. In lightof these emerging findings, in the near future established techniques may also be potentially usefulfor isolating skeletal proteins from similar marine calcifiers for drug discovery. As a discovery-drivenscience, the techniques discussed here allow researcher to identify candidate proteins for drugdiscovery and identify unknowns without missing unanticipated interactions. These techniquescan be employed to dramatically improve the range of applications within the field of marine drugdiscovery. Since the marine realm consists of diverse ecosystems and matrices in which these proteinsreside, the development of effective methods for accessing proteins will be a continuing challenge infuture years.

Acknowledgments: The author thanks Jochen Halfar for providing coralline samples. The author also thanksSteven Short for providing electrophoresis apparatus.

Conflicts of Interest: The author declares no conflict of interest.

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